title: Improved production of 2,3-butanediol in Bacillus amyloliquefaciens by over-expression of glyceraldehyde-3-phosphate dehydrogenase and 2,3-butanediol dehydrogenase.
abstract: BACKGROUND
  Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.
  
  
  RESULTS
  In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD(+) to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD(+). In this study, to improve 2,3-BD production, we first over-produced NAD(+)-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.
  
  
  CONCLUSIONS
  Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
authors.forename: Taowei
authors.initials: T
authors.lastname: Yang
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
authors.forename: Shang-Tian
authors.initials: ST
authors.lastname: Yang
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001407:Bacillus / Q000235:genetics* / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D002072:Butylene Glycols / Q000378:metabolism*; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics* / Q000378:metabolism; D053858:Metabolic Networks and Pathways
chemical_list: D002072:Butylene Glycols; D004163:Diphosphoglyceric Acids; C015891:glycerate 1,3-biphosphate; C026978:2,3-butylene glycol; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D005947:Glucose